A. Field of the Invention
This invention relates to a method for sequential analysis of polypeptides and proteins, and, more specifically, a photochemical method for binding polypeptides and proteins to a carrier in order to facilitate sequential analysis.
B. Prior Art
Recent advances in medical and pharmaceutical technology have uncovered a wide range of therapeutic uses for short- and long-chain polypeptide molecules. These substances, which are composed of a chain of linked amino acid molecules, control or participate in virtually all phases of cellular activity and structure. Direct control over specific metabolic levels or molecular characteristics of physiologically active polypeptides has been employed to achieve highly localized treatment of a variety of disorders, as well as promotion of desirable traits in commercial livestock. However, despite the significant potential for beneficial use of biologically active polypeptides, their size and structural complexity greatly limit the ability of scientists to understand and predict behavior in living systems.
A basic starting point for analysis of any linear chain polypeptide is determination of the precise sequence of its individual amino acid units. Researchers currently employ a variety of sequencing methodologies, the most common being the Edman degradation. In this method, successive amino acids are removed from the end of the chain by reacting the N-terminal amino acid residue with a reagent which allows selective removal of that residue. The resulting amino acid derivative is converted into a stable compound which can be chemically removed from the reaction mixture and identified.
Edman degradation and similar sequencing methods are commonly performed on a polypeptide immobilized on a solid support. This method of facilitating iterative amino acid cleavage offers a number of advantages. Assuming covalent bonding of the sample molecules to the carrier, virtually all of the sample will remain bound notwithstanding application of organic and aqueous solvents, salts or detergents. Hence, it is possible to achieve accurate sequence analysis even at picomole polypeptide levels. Furthermore, once attachment of the polypeptide has taken place, the Edman process will generally take place with a high degree of reliability.
After the sample has been bound to the solid support, the various Edman reagents are employed to cleave individual amino acid residues from the bound molecules for subsequent identification. With each repeated application of the reagents, the next N-terminal amino acid residue is removed. This process may be continued until an amino acid attached to the carrier is reached; although an attached amino acid can be cleaved from the next amino acid, it cannot be removed from the carrier for analysis. If additional amino acids are bound to the carrier, the sequence of amino acids residing between points of attachment may be cleaved and identified. However, when no further points of attachment exist, the unbound portion of the molecule enters solution and is thereby rendered unavailable for analysis. Hence, although multiple bonding sites impede analysis of the bound amino acid residues, a greater number of amino acids in the sample molecule will be available for sequencing than in the case of a single, early point of attachment. If attachment is random, one encounters a tradeoff between the absolute number of amino acid residues that may be successfully identified and the length of the peptide chain that will be available for analysis (see FIG. 2).
If bonding is not random, however, sensitivity to the favored amino acids will be decreased on a consistent basis. Nonrandom bonding will inhibit identification not only of the bound amino acid residues themselves, but also the terminal chain following the last favored amino acid. Random bonding assures a mixture of bound species, thereby disfavoring consistent loss of specific amino acids and a particular chain segment. Current immobilization procedures depend on specific amino acid reactivities, and therefore tend to inhibit random bonding. A general review of the present art is given in Laursen & Horn, Coupling Methods and Strategies in Solid-Phase Sequencing, in 25 Molecular Biology, Biochemistry and Biophysics 21 (S. Needleman, Ed., 1977).
Another limitation of many current immobilizing techniques is their utilization of carriers with functional groups that actively bond with sample molecules only within a specific pH range. This property curtails the range of polypeptides which may be analyzed, since polypeptides themselves require particular pH conditions to enter solution.
The ability of certain carriers to bond to polypeptides through photochemical means has been known for some time (see, e.g., U.S. Pat. No. 4,039,413). Because of the reactivity of the activated species, successful bonding does not require a particular pH environment. However, the procedure described in the aforementioned patent is limited by degradation of the carrier macromolecule's functional groups as a consequence of irradiation, appears to favor particular amino acids, and is not well-suited to multi-point attachment.